Lyme disease, the most commonly reported arthropod-borne disease in the United States, is caused by infection with the spirochete, Borrelia burgdorferi. Maintenance of B. burgdorferi in nature requires its acquisition from an infected mammalian host by an arthropod vector, growth and maintenance of the spirochete in that vector, and transmission from the infected vector to a mammalian host. Studies indicate that B. burgdorferi modulates its gene expression in response to changes in the vector and mammalian environments and expresion of proteins regulated in this maner play an important role in the B. burgdorferi life cycle and pathogenesis. The long-term objective of this project is characterization of genes and/or proteins that are crucial to maintenance of B. burgdorferi in the tick vector and its transmission to, and dissemination in, the mammalian host. We have employed functional and comparative genomics to identify B. burgdorferi genes whose expression is regulated during these processes and to define members of the RpoS and Rrp1 regulons. These investigations highlighted the fact that analysis of gene expression in the in vivo environment provides information that cannot be obtained by study under in vitro conditions designed to mimic the in vivo milieu. The results from these global regulatory studies have informed our decisions regarding genes/proteins that warrant further study. The selected genes display unique expression patterns in the tick and thus can serve as paradigms for the study of gene regulation in the vector. Specific aims 1 and 2 will involve a combination of detailed expression analyses and mutagenesis studies aimed at characterization of the expression and regulation for: 1) BBA74, an RpoS-regulated tick-phase protein with a unique expression profile during spirochete colonization of the vector; 2) the Glp operon that encodes proteins required for glycerol uptake and utilization, a process likely to be particularly important during B. burgdorferi growth and maintenance in ticks. In specific aim 3 we will perform global transcriptome analysis of B. burgdorferi during different developmental stages of the tick vector and elucidate the RpoS, Rrp1 and BosR regulons in ticks using a newly developed methodology that allows global assessment of B. burgdorferi gene expression from in vivo-derived transcripts. These studies will provide new insights into the natural history of B. burgdorferi in the tick vector that, in turn, may have implications for prevention and treatment of human Lyme disease. PUBLIC HEALTH RELEVANCE: Lyme disease, the most commonly reported tick-borne disease in the United States, is caused by infection with the spirochete, Borrelia burgdorferi. The findings obtained during the previous grant period have opened several new avenues of investigation that promise to reveal important aspects of B. burgdorferi gene regulation and processes at work in the tick environment. The combination of functional genomics and genetic approaches proposed here should illuminate important events that occur at the vector- host interface. This will provide new insights into the natural history of B. burgdorferi infection in nature which, in turn, may have implications for prevention and treatment of human Lyme disease.